Genetically engineered cell lines and systems for propagating varicella zoster virus and methods of use thereof

ABSTRACT

The present invention provides genetically engineered cell lines, recombinant vectors, and vaccines. The present invention also provides methods for generating an in vitro system for  Varicella zoster  virus (VZV), and the in vitro systems generated by these methods. The present invention further provides methods for reactivating VZV, and VZV reactivated by these methods. Finally, the present invention provides a method of screening for an agent for treating VZV infection.

RELATED APPLICATION

This application is a Divisional of prior Application U.S. Ser. No.10/436,706 filed May 12, 2003, which claims the benefit of U.S.Provisional Application No. 60/379,819, filed May 10, 2002.

BACKGROUND OF THE INVENTION

Varicella zoster virus (VZV) is the cause of chickenpox (varicella) andshingles (zoster). Varicella is a primary infection in which VZV infectsa naive host. Zoster is the result of the reactivation of VZV, which hasremained latent in its host, often for many years. A major paradox hasimpeded research on VZV for many years: VZV is highly infectious andspreads readily from an infected host to susceptible individuals, yet,it is extremely difficult to propagate in vitro, because it spreads onlyby direct cell-to-cell contact when it is grown in tissue culture. Thedissemination of VZV among a population of susceptible subjects ismediated by cell-free virions, which are thought to be airborne.Cell-to-cell spread of VZV in vitro does not depend on cell-freevirions, which are not released in viable form by infected cells intissue culture. Instead, cells that are infected in vitro fuse withtheir uninfected neighbors enabling infection to be transferredintracellularly.

SUMMARY OF THE INVENTION

The present invention provides a genetically engineered cell line stablytransformed with a nucleotide sequence encoding at least two full-lengthmannose-6-phosphate receptors.

Additionally, the present invention provides a genetically engineeredcell line stably transformed with a nucleotide sequence encoding afull-length mannose-6-phosphate receptor and a full-length insulin-likegrowth factor receptor.

Also provided is a recombinant vector comprising a nucleotide sequenceencoding at least two full-length mannose-6-phosphate receptors.

The present invention further provides a vaccine comprising anattenuated live virus produced by culturing genetically-engineered cellsstably transformed with a nucleotide sequence encoding at least twofull-length mannose-6-phosphate receptors and a pharmaceuticallyacceptable carrier.

The present invention is also directed to a method for generating an invitro system for Varicella zoster virus (VZV), by: (a) isolating entericganglia from guinea pig; and (b) contacting the enteric ganglia withcell-free VZV to generate latent expression of the VZV. Also provided isan in vitro system for VZV generated by this method.

The present invention further provides an in vitro system for Varicellazoster virus (VZV), comprising an enteric ganglion that has beencontacted with cell-free VZV to produce a latent VZV, wherein the VZV issubsequently reactivated to express VZV in an active form.

Additionally, the present invention provides a method for reactivatingVaricella zoster virus (VZV), by: (a) isolating enteric ganglia fromguinea pig; (b) contacting the enteric ganglia with cell-free VZV togenerate latent expression of the VZV; and (c) contacting the infectedganglia with a vector containing a nucleic acid sequence encoding VZVORF61 or a homologue thereof. Also provided is a Varicella zoster virusreactivated by this method.

Finally, the present invention provides a method of screening for anagent for treating Varicella zoster virus (VZV) infection, comprisinguse of an in vitro system for VZV, wherein the in vitro system comprisesan enteric ganglion that has been contacted with cell-free VZV toproduce a latent VZV, and wherein the VZV is subsequently reactivated toexpress VZV in an active form.

Additional aspects of the present invention will be apparent in view ofthe description which follows.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is an illustration of a Varicella zoster Virus (VZV) particle;

FIG. 2 illustrates that mannose-6-phosphate receptors (MPRs) sortlysosomal enzymes and target them to endosomes;

FIG. 3 illustrates intracellular transport of VZV and packing of thefinal envelope in the trans-Golgi network (TGN);

FIG. 4 illustrates that MPRs direct newly assembled VZV to lateendosomes;

FIG. 5 illustrates the mechanism by which VZV particles receive theirfinal envelope in the TGN;

FIG. 6 demonstrates that human embryonic lung cells (HELF) infection bycell-free VZV is inhibited by phosphorylated sugars;

FIG. 7 demonstrates that expression of cation-independent MPRs(MPR^(ci)s) in MeWo cells is downregulated by antisense cDNA;W

FIG. 8 shows that downregulation of MPR^(ci)s inhibits infection bycell-free VZV;

FIG. 9 demonstrates that MPR^(ci)-KO (knock-out) cells can be infectedby cell-associated VZV;

FIG. 10 illustrates that MPR^(ci)-KO cells release infectious VZV intothe culture supernatant;

FIG. 11 illustrates that VZV nucleocapsids assemble in the nuclei ofinfected KO-MeWo cells;

FIG. 12 shows that enveloped VZV enters the perinuclear cisterna ofKO-MeWo cells;

FIG. 13 demonstrates that, in KO-MeWo cells, transport vesicles containindividual virions;

FIG. 14 demonstrates that VZV accumulates in late endosomes in parentalMeWo cells;

FIG. 15 illustrates that enveloped VZV is released intact in thesuperficial epidermis;

FIG. 16 shows that lytic infection of neurons occurs when non-neuronalcells are present;

FIG. 17 illustrates that neurons die within two days when infection islytic;

FIG. 18 demonstrates that expression of HSV ICP0 causes VZV toreactivate in enteric neurons;

FIG. 19 shows that latent VZV reactivates in enteric neurons forced toexpress HSV ICP0;

FIG. 20 shows that cultured ganglia contain few non-neuronal cells;

FIG. 21 demonstrates that RT-PCR reveals expression of VZV DNA and RNAin isolated ganglia;

FIG. 22 shows that infected ganglia contain mRNA and DNA encoding VZVproteins;

FIG. 23 illustrates that ORF29 mRNA is found by in situ hybridization insubsets of neurons;

FIG. 24 demonstrates that infected neurons are ORF29p− but notgE-immunoreactive;

FIG. 25 shows that ORF62p, 4p, and 21p are present in the cytoplasm ofinfected neurons.

DETAILED DESCRIPTION OF THE INVENTION

The inventors have demonstrated that viable VZV is not released to themedium by cultured cells because newly assembled virions are divertedfrom the secretory pathway to late endosomes where they encounter anacidic environment and lysosomal enzymes. The viral particles are thendegraded in these endosomes before the virions are released to themedium by exocytosis. Only degraded VZV, which is not infectious, thusis delivered to the extracellular medium. VZV receives its finalenvelope in the trans-Golgi network (TGN). This cellular organelle is anintracellular sorting location and is the site where lysosomal enzymesare separated from proteins destined to be secreted. Diversion oflysosomal enzymes occurs because they contain mannose-6-phosphate (Man6-P) groups that enable them to bind to mannose-6-phosphate receptors(MPRs), which are responsible for routing the lysosomal enzymes toendosomes. There are 2 MPRs, a large cation-independent molecule, whichis also the receptor for insulin-like growth factor 2 (Man 6-P/IGF2R),and a small cation-dependent molecule. Lysosomal enzymes dissociate fromMPRs in endosomes and are transported within the interiors of vesiclesto lysosomes, while the MPRs are transported in membranes either back tothe TGN or to the plasma membrane. The Man 6-P/IGF2Rs in the plasmamembrane are able to bind to extracellular molecules that express Man6-P groups, such as those of lysosomal enzymes, and mediate theendocytosis of these ligands.

The inventors surprisingly found that newly assembled VZV is divertedfrom the secretory pathway of infected cells by Man 6-P/IGF2Rs, which isthus responsible for the delivery of enveloped virions to endosomes.This is supported by observations that glycoproteins of the VZV envelopecontain Man 6-P groups. In addition, the simple addition of Man 6-P tothe medium in which cells are growing prevents these cells from becominginfected by cell-free VZV. Man 6-P interferes with the interactionbetween plasma membrane Man 6-P/IGF2R and the Man 6-P groups ofextracellular molecules. Man 6-P does not prevent the cell-to-cellspread of VZV in vitro, which because it depends on the fusion ofadjacent cells, which is independent of the interaction of viral Man 6-Pwith Man 6-P/IGF2R. These observations suggest that the Man 6-P groupsof the glycoproteins of the VZV envelope interact with cellular Man6-P/IGF2Rs and thus play critical roles in the ability of cell-free VZVto infect target cells and in the transport within infected cells ofnewly assembled VZV from the TGN to endosomes.

Accordingly, the inventors produced a cell line that is deficient in Man6-P/IGF2R. Because of the strong preference of VZV for human cells, theMan 6-P/IGF2R-deficient cell line was derived from MeWo cells, a humanmelanoma tumor cell line, which is known to be susceptible to infectionby VZV and to support the growth of VZV in vitro.

The Man 6-P/IGF2R-deficient MeWo cells were found to be far moreresistant to infection by cell-free VZV than their parental control MeWocells; however, infected cells can transfer VZV infection to the Man6-P/IGF2R-deficient MeWo cells as readily as to the parental controlMeWo cells. The medium in which parental control MeWo cells is growingis not infectious and cannot be used to transfer infection with VZV tosusceptible target cells (human embryonic lung cells [HELF] are employedas the susceptible targets). In contrast, the medium in which Man6-P/IGF2R-deficient MeWo cells is growing does contain infectious VZVand can be used to transfer infection with VZV to target HELF cells.This is the first cell line that, when infected with VZV, releasessubstantial quantities of infectious VZV particles to the ambientmedium. The medium in which the Man 6-P/IGF2R-deficient MeWo cells aregrowing is thus extraordinarily useful as a source of intact,non-degraded, infectious VZV. The resistance of the Man6-P/IGF2R-deficient MeWo cells to infection by cell-free VZV confirmsthat the Man 6-P/IGF2R plays an important role in enabling VZV to entertarget cells. Infection of Man 6-P/IGF2R-deficient MeWo cells must beaccomplished by adding other infected cells to them. The infected cellscan thus infect the Man 6-P/IGF2R-deficient MeWo cells by cell-to-cellcontact, which is Man 6-P/IGF2R-independent.

The successful maintenance and easy use of the Man 6-P/IGF2R-deficientMeWo cells depends on the knockdown of the Man 6-P/IGF2R beingincomplete. When the Man 6-P/IGF2R expression is abolished, theresulting cells grow poorly and slowly. The current Man6-P/IGF2R-deficient MeWo cells express small amounts of the Man6-P/IGF2R and thus grow adequately, can be maintained without excessdifficulty, yet still release infectious VZV.

Uses of the Man 6-P/IGF2R-deficient MeWo cells include the developmentof new and reliable methods to produce the VZV vaccine. This vaccine isan attenuated live virus that must be produced by cultured cells. Itgrows in culture, like wild-type VZV, only by cell-to-cell contact. Thefinal product has to be liberated from the small intracellularcompartment that contains newly assembled virions that have not yet beentransported to endosomes. Virions that have been degraded in endosomesare useless as components of the vaccine. Yields of live virus are thusvery low. Propagation in the Man 6-P/IGF2R-deficient MeWo cells shouldproduce high yields of much more readily purified viable virus. Inaddition to being extremely useful in vaccine production, the Man6-P/IGF2R-deficient MeWo cells should be just as helpful in developingnew strains of VZV for future vaccines. Finally, the virus produced inMan 6-P/IGF2R-deficient MeWo cells is likely to be much more uniform andreadily controlled than that produced in ordinary tissue culture cells.The non-controllable degradation of virions is avoided, yields areimproved, and a step of cell lysis can be avoided in vaccine production.Contamination of the vaccine with cellular constituents and otherviruses that might be contained in tissue culture cells can also beminimized.

The Man 6-P/IGF2R-deficient MeWo cells will also help in research on VZVitself. As such, Man 6-P/IGF2R-deficient MeWo cells will be of greatvalue and sought after by research workers hoping to develop antiviraldrugs and those seeking to understand the basic properties of VZV.Although the Man 6-P/IGF2R has been most strongly implicated asimportant, as described in the biology of VZV, the receptor may alsoplay roles in the biology of other related herpesviruses, such as herpessimplex virus types 1 and 2 and pseudorabies virus. These virions dospread through media in vitro, but they may well spread more readilywhen propagated in Man 6-P/IGF2R-deficient MeWo cells. The Man6-P/IGF2R-deficient MeWo cells, finally, are likely to be attractive tocell biologists who are interested in the basic properties of lysosomes,endosomes, receptor-mediated endocytosis, autophagy, and thepathogenesis of lysosomal storage disease.

The present invention is described in the following Examples, which areset forth to aid in the understanding of the invention, and should notbe construed to limit in any way the scope of the invention as definedin the claims which follow thereafter.

EXAMPLES Example 1 Production of Man 6-P/IGF2R-Deficient Cells

The deficient expression of Man 6-P/IGF2Rs was induced by stablytransfecting parent MeWo cells (available from the European Collectionof Cell Cultures (ECACC) Salisbury, Wiltshire) with cDNA encoding thefull-length Man 6-P/IGF2R in the antisense configuration. The cDNAconstruct was packaged in a retroviral vector that also contained apuromycin resistance gene. The cells that were infected by the vector,and thus expressed mRNA encoding the Man 6-P/IGF2R in the antisenseconfiguration, was selected in media containing puromycin. The successof the knockdown of the Man 6-P/IGF2R was confirmed by Western analysisand by immunocytochemistry; both techniques demonstrated reduced Man6-P/IGF2R expression. In addition, the Man 6-P/IGF2R-deficient MeWocells were found to contain less cathepsin D (a lysosomal enzyme thatwas investigated as a marker) than control parental MeWo cells. Thisdemonstration was again accomplished by Western analysis and byimmunocytochemistry. The Man 6-P/IGF2R-deficient cells also secreted agreater amount of another lysosomal enzyme, acid phosphatase, thanparental MeWo cells (shown by direct measurement of acid phosphataseactivity in cells and in media and by Western analyses of media and celllysates).

The foregoing results are summarized in FIGS. 1-15. The data confirmthat the Man 6-P/IGF2R-deficient MeWo cells have a reduced ability todivert lysosomal enzymes from their secretory pathway to endosomes.

Example 2 Development of Animal Model of VZV Latency

VZV has a narrow host range. However, it is known to be neurotropic, andto become latent in sensory ganglia. Because the enteric nervous system(ENS) contains intrinsic sensory neurons, the inventors postulated thatVZV may infect, and become latent in, the ENS (e.g., enteric neuronsand/or enteric ganglia).

The ENS is an independent autonomic division that is capable ofregulating the behavior of the gut without input from the centralnervous system (CNS). The ENS has independence from the CNS because itcontains two main types of intrinsic primary afferent neurons (IPANS):submucosal IPANS (containing cholinergic-CGRP or substance-P) ormyenteric IPANS (containing cholinergic calbindin). The inventors foundthat reactivation of VZV in enteric neurons could provide a source ofvisceral zoster, and present a model for understanding the origin ofzoster. This was established by first testing the ability of VZV toestablish latency in guinea pig enteric ganglia in vitro, and then bytesting the ability of the latent VZV to reactivate.

The inventors first isolated ganglia from guinea pig small intestine.The LM-MP was mechanically dissected from the bowel, and dissociatedwith collagenase. Myenteric ganglia then were individually selected andcultured. Mitotic inhibitors were used to decrease growth ofnon-neuronal cells, enabling enteric neurons to reorganize andinterconnect. Cell-free VZV was adsorbed for 4 h, approximately 5 daysafter the ganglia were plated. Cultures were maintained for 4-6 weeksthereafter.

As shown in FIGS. 16-25, VZV infected guinea pig enteric ganglia invitro. Latent infection occurred with the use of cultures highlyenriched in neurons. It was observed that neurons expressed only thoseVZV proteins that had been reported to be expressed during VZV latencyin human sensory ganglia (no glycoproteins). In this regard, it is notedthat ORF62p and ORF29p are cytoplasmic, not nuclear. Lytic infectionrequired the presence of non-neuronal cells. Both neurons andnon-neuronal cells expressed glycoproteins, and ORF62p and ORF29p werefound in nuclei. Reactivation occurred in neurons, and likely occurredin glia. Reactivation was induced by expression of HSV ICP0 (the HSVhomologue of VZV ORF61). Neurons expressed glycoproteins, and ORF29p wasfound in nuclei. The model of the in vitro reactivation of VZV describedherein is the first in vitro model for shingles.

While the foregoing invention has been described in some detail forpurposes of clarity and understanding, it will be appreciated by oneskilled in the art, from a reading of the disclosure, that variouschanges in form and detail can be made without departing from the truescope of the invention in the appended claims.

1. A vaccine comprising an attenuated live virus produced by culturinggenetically-engineered cells stably transformed with a nucleotidesequence encoding at least two full-length mannose-6-phosphate receptorsand a pharmaceutically acceptable carrier.
 2. A method for generating anin vitro system for Varicella zoster virus (VZV), comprising the stepsof: (a) isolating enteric ganglia from guinea pig; and (b) contactingthe enteric ganglia with cell-free VZV to generate latent expression ofthe VZV.
 3. An in vitro system for VZV generated by the method of claim2.
 4. The in vitro system of claim 3, wherein the method furthercomprises the step of reactivating the VZV by contacting the system witha vector containing a nucleic acid sequence coding for VZV ORF61 or ahomologue thereof.
 5. The in vitro system of claim 4, wherein thehomologue is HSV ICP0.
 6. An in vitro system for Varicella zoster virus(VZV), comprising an enteric ganglion that has been contacted withcell-free VZV to produce a latent VZV, wherein the VZV is subsequentlyreactivated to express VZV in an active form.
 7. A method forreactivating Varicella zoster virus (VZV), comprising the steps of: (a)isolating enteric ganglia from guinea pig; (b) contacting the entericganglia with cell-free VZV to generate latent expression of the VZV; and(c) contacting the infected ganglia with a vector containing a nucleicacid sequence encoding VZV ORF61 or a homologue thereof.
 8. The methodof claim 7, wherein the homologue is HSV ICP0.
 9. A Varicella zostervirus reactivated by the method of claim
 10. 10. A method of screeningfor an agent for treating Varicella zoster virus (VZV) infection,comprising use of an in vitro system for VZV, wherein the in vitrosystem comprises an enteric ganglion that has been contacted withcell-free VZV to produce a latent VZV, and wherein the VZV issubsequently reactivated to express VZV in an active form.